Antibody vaccine conjugates and uses therefor

ABSTRACT

The present invention provides novel antibody vaccine conjugates and methods of using the same to induce a cytotoxic T cell (CTL) response. In a particular, embodiment, the vaccine conjugate includes a human chorionic gonadotropin beta subunit (βhCG) antigen linked to an anti-mannose receptor (MR) antibody.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/903,191 (filed Jul. 30, 2004), entitled “Antibody Vaccine Conjugatesand Uses Therefor”, which is a continuation-in-part application of U.S.application Ser. No. 10/769,144 (filed Jan. 30, 2004), entitled“Antibody Vaccine Conjugates and Uses Therefor”, which claims priorityto U.S. Provisional Patent Application No. 60/443,979 (filed Jan. 31,2003), entitled “Antibody Vaccine Conjugates and Uses Therefor”. Thecontents of each of these applications is incorporated herein byreference.

BACKGROUND OF THE INVENTION

The immune response is initiated at the level of professional antigenpresenting cells (APC), which include dendritic cells (DC) andmacrophages (Mg), that reside in tissues throughout the body. DCsexpress high levels of cell surface molecules and complementaryreceptors that interact with T lymphocytes and, therefore, induce potentimmune responses. DCs also secrete cytokines, chemokines and proteaseswhich initiate immune responses and culminate in the amplification ofboth cellular and humoral immunity.

DCs express on their surface major histocompatibility complex (MHC)molecules that bind fragments of antigens. T cells which express T cellreceptors (TCR) that recognize such antigen-MHC complexes becomeactivated and initiate the immune cascade. In general, there are twotypes of MHC molecules, MHC class I and MHC class II molecules. MHCclass I molecules present antigen to specific CD8⁺ T cells and MHC classII molecules present antigen to specific CD4⁺ T cells.

For effective treatment of many diseases, particularly cancers, vaccinesmust elicit a potent cytotoxic T lymphocyte (CTL) response, alsoreferred to as a cytotoxic T cell response. Cytotoxic T cellspredominantly include CD8⁺ T cells which recognize antigen in thecontext of MHC class I. The processing of antigens in the context of MHCclass I molecules differs significantly from that of MHC class IImolecules. Antigens delivered exogenously to APCs are processedprimarily for association with MHC class II molecules. In contrast, dueto the intracellular location of MHC class I molecules, antigensdelivered endogenously to APCs are processed primarily for associationwith MHC class I molecules. This is not only true for APCs, as allnucleated cells express MHC class I molecules, and are continuouslydisplaying on their surface endogenously produced antigens inassociation with MHC class I molecules.

For this reason, cells infected with virus or tumor cells expressingunique proteins can be targeted by CTLs when viral or tumor antigens aredisplayed as a peptide bound to MHC class I molecules. However, DCs,under specific conditions, have the unique capacity also to allowexogenous antigens access to internal compartments for binding to MHCclass I molecules, so that they are presented to T cells via both MHCclass I and class II pathways. This process is called cross-priming orcross-presentation.

Accordingly, while antibody-mediated responses have demonstratedimpressive protective or therapeutic efficacy for specific diseases whendirected against particular secreted or cell surface antigens, the mosteffective immunotherapy for many diseases appears to require Tcell-mediated immune responses, particularly CTL responses. Sinceeffective CTL responses are not limited to extracellular antigens, thereexist possibilities for developing antigen-based therapeutic vaccinesthat are not effective antibody targets. Therefore, new methods forgenerating CTLs in response to disease-associated antigens have been ofgreat interest, as these cells are thought to be critical for theefficacy of many vaccines in general, and essential to most therapeuticcancer vaccines.

One vaccine approach which has been tested to date employs immunizingwith antigenic peptides. This method of immunization bypasses the needfor antigen uptake and processing and relies on the ability of thepeptide to bind directly to MHC class I molecules already expressed onthe surface of the APC. Although this method has clearly shown evidenceof CTL induction in patients, the method has several limitations. Theantigenic peptide must be pre-established, different peptides arerequired for individuals with different MHC haplotypes, and peptides areshort-lived in vivo.

Another approach which has been tested employs antibody-antigencomplexes. Paul et al. (62) showed that antibodies specific for a givenantigen could enhance humoral immune responses against the antigen inmice, presumably by delivering the immune complexes to Fc receptors forIgG (FcγR) expressed on APCs. Wernersson and colleagues (63) studied therole of individual FcγRs in the enhancement of immune responses usingimmune complexes in vivo. Their studies demonstrated that FcγRI issufficient to mediate enhanced immune responses. However, such immunecomplexes do not target APCs specifically, as they also bind to Fcreceptors on many cells that are not involved in antigen presentation,thereby, decreasing the efficiency of antigen delivery.

Subsequent studies have used antibodies to selectively target antigensto a variety of receptors on APCs, and have demonstrated that suchselective delivery is capable of inducing humoral responses (66,67). Inaddition, it has been shown that immune complexes bound to FcR on DCsare processed and presented in context of MHC class I (64,65). Moreover,many such FcR-targeting approaches are limited because FcR are expressedon many non-APC such as platelets and neutrophils. Ideally, a vaccinethat targets APC specifically and is capable of inducing an effectiveMHC class I-restricted CTL response, as well as an effective MHC classII-restricted TH response could offer improved efficacy in treatingcertain diseases.

Similarly, mannosylated antigens have been shown to induce humoralimmune responses and T cell-mediated immune responses, such as CTLresponses. However, mannosylated antigens do not target APC specificallydue to the significant abundance of other mannose binding proteins.Furthermore, mannosylated proteins are internalized by immature DCsthrough macropinocytic mechanisms. Therefore, the mechanisms and natureof immune responses generated by mannosylation of antigens differsgreatly from that generated by specific targeting of antigens to mannosereceptors using antibodies.

Since current methods do not efficiently and specifically target APCs,many therapeutic vaccines require the purification of DC from patients,which are reinfused after exposure to the antigen.

Accordingly, the need exists for improved vaccines capable ofefficiently targeting APCs and generating antigen-specific Tcell-mediated immune responses, including antigen-specific CTLresponses, required for effective treatment of many diseases.

SUMMARY OF THE INVENTION

The present invention provides antibody-based vaccines and methods forgenerating antigen-specific T cell-mediated immune responses requiredfor effective treatment of many diseases. In particular, a potentantigen-specific cytotoxic T lymphocyte (CTL) response is induced bytargeting one or more protein antigens to antigen presenting cells(APCs), using antibodies which bind to particular receptors expressed onAPCs. Preferred receptors include C-lectins, particularly the humanmannose receptor, which are expressed on both dendritic cells (DCs) andmacrophages. As demonstrated by way of the present invention, targetingthe mannose receptor using antibody-antigen conjugates results inprocessing of the antigen through both MHC class I and class IIpathways. Thus, antigen-specific CTLs (e.g., CD8⁺ T cells) are induced,as well as other important effector T cells, including helper T cells(e.g., CD4⁺ T cells). Accordingly, in one aspect, the present inventionprovides a method for inducing or enhancing a CTL response against anantigen by forming a conjugate of the antigen and a monoclonal antibodywhich binds to a human APC, e.g., a monoclonal antibody which binds tothe human mannose receptor expressed on human APC. The conjugate is thencontacted, either in vivo or ex vivo, with APCs such that the antigen isinternalized, processed and presented to T cells in a manner whichinduces or enhances a CTL response (e.g., a response mediated by CD8⁺cytotoxic T cells) against the antigen. In a preferred embodiment, thisserves also to induce a helper T cell response (e.g., a responsemediated by CD4⁺ helper T cells) against the antigen. Thus, the immuneresponse is induced through both MHC class I and MHC class II pathways.The APCs can also be contacted with an adjuvant or an immunostimulatoryagent, such as a cytokine, which stimulates proliferation of dendriticcells to further enhance the immune response. Such agents can becontacted separately from the molecular conjugate or linked to themolecular conjugate.

Accordingly, in a particular embodiment, the invention provides avaccine conjugate comprising a monoclonal antibody which binds to ahuman APC linked to an antigen and to an immunostimulatory agent. Theimmunostimulatory agent can be linked to the conjugate either covalentlyor non-covalently. Alternatively, the immunostimulatory agent can begenetically fused to the conjugate (e.g., expressed together with theconjugate as a single fusion protein). A variety of suitableimmunostimulatory agents can be employed including, but not limited to:CD40 ligand; cytokines, such as IFN-α, IFN-β, IFN-γ and IL-2;colony-stimulating factors, such as G-CSF (granulocytecolony-stimulating factor) and GM-CSF (granulocyte-macrophagecolony-stimulating factor); an anti-CTLA-4 antibody; toll receptoragonists (e.g., flagellin and MALP-2 (macrophage activatinglipopeptide-2); LPS (endotoxin); R837 (3M Pharmaceuticals, St. Paul,Minn.); R848 (3M Pharmaceuticals, St. Paul, Minn.); polyI:C(inosine:cytosine polynucleotide); ssRNA; dsRNA; Bacille Calmette-Guerin(BCG); Levamisole hydrochloride; and intravenous immune globulins.

A variety of suitable antibodies can be employed in the conjugates ofthe present invention including, but not limited to those derived fromany species (e.g., human, murine, rabbit etc.) and/or those engineeredand expressed recombinantly (e.g., chimeric, humanized and humanantibodies). Preferred antibodies include human monoclonal antibodies.Antibodies used in the invention also can include any antibody isotype,such as IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, or IgE,although preferred antibodies are of the IgG isotype. The antibodies canbe whole antibodies or antigen-binding fragments thereof including, forexample, Fab, F(ab′)₂, Fv and single chain Fv fragments.

Preferred antibodies for use in the present invention include humanmonoclonal antibodies that bind to the human mannose receptor. In oneembodiment, the antibody is encoded by human heavy chain and human kappalight chain nucleic acids comprising nucleotide sequences in theirvariable regions as set forth in SEQ ID NO:3 and SEQ ID NO:7,respectively, or a nucleotide sequence that is sufficiently homologousto SEQ ID NO:3 or SEQ ID NO:7 such that the antibody retains the abilityto bind to dendritic cells.

Still other preferred human antibodies include those characterized asbinding to the human mannose receptor and having a human heavy chain andhuman kappa light chain variable regions comprising the amino acidsequences as set forth in SEQ ID NO:4 and SEQ ID NO:8, respectively, oran amino acid sequence that is sufficiently homologous to SEQ ID NO:4 orSEQ ID NO:8 such that the antibody retains the ability to bind todendritic cells.

Still other particular human antibodies of the invention include thosewhich comprise a complementarity determining region (CDR) domain havinga human heavy and light chain CDR1 region, a human heavy and light chainCDR2 region, and a human heavy and light chain CDR3 region, wherein

(a) the CDR1, CDR2, and CDR3 of the human heavy chain regions comprisean amino acid sequence selected from the group consisting of the aminoacid sequences of the CDR1, CDR2, and CDR3 regions shown in FIG. 8 (SEQID NOs:13, 14, or 15), and conservative sequence modifications thereof,and

(b) the CDR1, CDR2, and CDR3 of the human light chain regions comprisean amino acid sequence selected from the group consisting of the aminoacid sequences of the CDR1, CDR2, and CDR3 regions shown in FIG. 9 (SEQID NOs:16, 17, or 18), and conservative sequence modifications thereof.

Antibodies derived from a particular germline sequence, for example,antibodies obtained from a system using human immunoglobulin sequences,e.g., by immunizing a transgenic mouse carrying human immunoglobulingenes or by screening a human immunoglobulin gene library, are alsoincluded in the present invention.

Human antibodies for use in the invention can be produced recombinantlyin a host cell, such as a transfectoma (e.g., a transfectoma consistingof immortalized CHO cells or lymphocytic cells) containing nucleic acidsencoding the heavy and light chains of the antibody, or be obtaineddirectly from a hybridoma which expresses the antibody (e.g., whichincludes a B cell obtained from a transgenic nonhuman animal, e.g., atransgenic mouse, having a genome comprising a human heavy chaintransgene and a human light chain transgene that encode the antibody,fused to an immortalized cell). In a particular embodiment, theantibodies are produced by a hybridoma, or by a host cell (e.g., a CHOcell) transfectoma containing human heavy chain and human light chainnucleic acids which comprise nucleotide sequences SEQ ID NOs:3 and 7,respectively, and conservative modifications thereof.

Suitable antigens for use in the present invention include any antigen,or antigenic portion thereof, against which a protective or therapeuticimmune responses is desired including, for example, a variety of tumorand infectious disease antigens. Particular antigens can be selectedfrom, among others, human chorionic gonadotropin beta subunit (βhCG),Gp100, prostate associated antigen (PSA), Pmel-17, colon, lung,pancreas, breast, ovary, and germ cell derived tumor cell antigens,viral proteins, bacterial proteins, carbohydrates, and fungal proteins.In accordance with the invention, such antigens are linked to antibodiesto form highly effective antibody vaccine conjugates.

In another aspect, the present invention provides a particular antibodyvaccine conjugate that includes βhCG linked to an antibody which bindsto the human mannose receptor. In one embodiment, the conjugatecomprises a human heavy chain which is linked to βhCG, such as theB11-βhCG conjugate described herein having a heavy chain comprising theamino acid sequence shown in SEQ ID NO:10. A single chain version of theB11-βhCG conjugate is also provided, comprising the amino acid sequenceshown in SEQ ID NO: 12.

The present invention further provides compositions (e.g.,pharmaceutical compositions) containing one or more antibody vaccineconjugates of the invention. The compositions can additionally includeone or more adjuvants or other agents known to enhance immune responsesand/or increase the activity of APCs.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a map of the molecular conjugate (SEQ ID NOs:11 and 12)encoding a fusion protein containing the single chain B11 antibodylinked to βhCG antigen (pB11sfv-βhCG).

FIG. 2 shows a map of the molecular conjugate (SEQ ID NOs:9 and 10)encoding a fusion protein containing the whole B11 antibody linked toβhCG antigen (βhCG-B11 construct).

FIG. 3 is a schematic illustration of a molecular conjugate. The antigenis genetically fused to the heavy chains of the intact antibody.

FIG. 4 is a graph based on flow cytometry studies which shows that theβhCG-B11 construct binds specifically to cultured human DC expressingMR.

FIG. 5 is a graph showing that the βhCG-B11 construct inducesβhCG-specific cytotoxic T cells.

FIG. 6 is a graph showing that the βhCG-B11 construct inducesβhCG-specific cytotoxic T cells.

FIG. 7 is a bar graph showing that the βhCG-B11 construct induces Thelper response.

FIG. 8 shows the nucleotide sequence (SEQ ID NO:3) and correspondingamino acid sequence (SEQ ID NO:4) of the heavy chain V region of humanmonoclonal antibody B11 with CDR regions designated (SEQ ID NOs: 13, 14,and 15).

FIG. 9 shows the nucleotide sequence (SEQ ID NO:7) and correspondingamino acid sequence (SEQ ID NO:8) of the light (kappa) chain V region ofhuman monoclonal antibody B11 with CDR regions designated (SEQ ID NOs:16, 17, and 18).

FIG. 10 is a diagram showing the predicted T cell epitopes of theβhCG-B11 construct as analyzed using web-based predictive algorithms(BIMAS & SYFPEITHI). T cell epitopes were found for potential binding toHLA-A2, HLA-B7 and HLA-DR molecules. Several epitopes were alsopredicted from the B11 segment of βhCG-B11. No T cell epitope wasidentified in the 37 aa long C-terminal peptide.

FIG. 11 is a graph showing CTL specific for the βhCG-B11 constructrecognize the scFv form of the antigen, B11 sfv-βhCG presented by DCs.

FIG. 12 shows the amino acid sequence (SEQ ID NO:4) of the heavy chain Vregion of human monoclonal antibody B11 compared to the germlinesequence (SEQ ID NO:30), VH5-51 germline.

FIG. 13 shows the nucleotide sequence (SEQ ID NO:3) of the heavy chain Vregion of human monoclonal antibody B11 compared to the germlinesequence (SEQ ID NO:29), VH5-51 germline.

FIG. 14 shows the amino acid sequence (SEQ ID NO:8) of the light (kappa)chain V region of human monoclonal antibody B11 with CDR regionsdesignated compared to the germline sequence (SEQ ID NO:32), Vk-L15germline.

FIG. 15 shows the nucleotide sequence (SEQ ID NO:7) of the light (kappa)chain V region of human monoclonal antibody B11 with CDR regionsdesignated compared to the germline sequence (SEQ ID NO:31), Vk-L15germline.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that important Tcell-mediated immune responses can be generated by targeting antigens toantigen presenting cells (APCs) using antibodies directed againstparticular cellular receptors. Specifically, for effective treatment ofmany diseases, such as cancers and infectious diseases, vaccines mustelicit a potent antigen-specific cytotoxic T lymphocyte (CTL) response,primarily mediated by CD8+ T cells which recognize antigen in thecontext of MHC class I. For optimal immunization, this is preferablyaccompanied by other important effector T cell functions, includinginduction of antigen-specific helper T cells, such as CD4+ T cells,which recognize antigen in the context of the MHC class II pathway.Thus, effective vaccines should induce antigen-specific CTLs, preferablyin combination with other T cell-mediated immune responses, throughmultiple MHC pathways.

Accordingly, the present invention provides novel antibody-based vaccineconjugates and methods for inducing or enhancing antigen-specificcytotoxic T cell (CTL) responses. Therapies of the invention employmolecular conjugates comprising antibodies which bind to antigenpresenting cells (APC), such as dendritic cells (DC) and macrophages,linked to an antigen. The molecular conjugates can be administered aloneor with other immunostimulatory agents and/or adjuvants that furtherenhance the immune response against the antigen. In one embodiment, theimmunostimulatory agent is co-administered with the molecular conjugate.In another embodiment, the immunostimulatory agent is administered priorto or after administration of the molecular conjugate. In yet anotherembodiment, the immunostimulatory agent is linked (e.g., covalently,non-covalently or recombinantly) to the molecular conjugate. Forexample, the immunostimulatory agent can be genetically fused orchemically linked to the molecular conjugate via, e.g., the heavy and/orlight chain of the antibody portion of the conjugate.

Antibodies which target APCs are known in the art and include, forexample, antibodies which target Class I or Class II majorhistocompatibility (MHC) determinants on APC (78, 79, 81, 83). Otherantibodies include those which target Fc receptors on APCs (77, 79, 80,81, 82, 83), as well as surface immunoglobulins on B cells (84).

In a particular embodiment exemplified herein, the molecular conjugateincludes an antibody which binds to the mannose receptor (MR) on humanDCs, linked to the βhCG antigen. Such conjugates can be contacted withAPCs either in vivo or ex vivo to generate desired CTL responses.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein, the term “antigen presenting cell (APC)” refers to aclass of immune cells capable of internalizing and processing anantigen, so that antigenic determinants are presented on the surface ofthe cell as MHC-associated complexes, in a manner capable of beingrecognized by the immune system (e.g., MHC class I restricted cytotoxicT lymphocytes and/or MHC class II restricted helper T lymphocytes). Thetwo requisite properties that allow a cell to function as an APC are theability to process endocytosed antigens and the expression of MHC geneproducts. Examples of APCs include dendritic cells (DC), mononuclearphagocytes (e.g., macrophages), B lymphocytes, Langerhans cells of theskin and, in humans, endothelial cells.

The term “dendritic cell (DC)” as used herein, includes immature andmature DCs and related myeloid progenitor cells that are capable ofdifferentiating into DCs or related antigen presenting cells (e.g.,monocytes and macrophages). DCs express high levels of cell surfacemolecules and complementary receptors that interact with T lymphocytes(e.g., C-type lectins, such as the mannose receptor) and, therefore, arecapable of inducing induce potent immune responses. DCs also secretecytokines, chemokines and proteases which initiate an immune responseand culminates in the amplification of both cellular and humoralimmunity. DCs also express on their surface major histocompatibiltycomplex (MHC) molecules that bind fragments of antigens. T cells whichrecognize these antigen-MHC complexes become activated and initiate theimmune cascade. In a preferred embodiment, binding of an antibodyportion of the molecular conjugate of the invention to a dendritic cellresults in internalization of the conjugate by the dendritic cell.

The term “macrophage mannose receptor” or “MR” refers to a member of afamily of C-type lectin receptors characterized by repeatedcarbohydrate-recognition domains (CRD) in the extracellular portion anda short cytoplasmic tail containing two putative clathrin targetingsequences (34,35,37). In addition, the MR contains N-terminal cysteinerich and fibronectin domains. The different domains of the mannosereceptor have specific binding capacity for various ligands includinglysosomal enzymes, micro-organisms, pituitary hormones,glycosoaminoglycans, and sulfated blood group antigens (38-40).

“MHC molecules” include two types of molecules, MHC class I and MHCclass II. MHC class I molecules present antigen to specific CD8⁺ T cellsand MHC class II molecules present antigen to specific CD4⁺ T cells.Antigens delivered exogenously to APCs are processed primarily forassociation with MHC class II. In contrast, antigens deliveredendogenously to APCs are processed primarily for association with MHCclass I. However, under specific conditions, DCs have the uniquecapacity to allow exogenous antigens access to internal compartments forbinding to MHC class I molecules, in addition to MHC class II molecules.This process is called “cross-priming” or “cross-presentation.”

As used herein, the term “immunostimulatory agent” refers to compoundscapable of stimulating APCs, such as DCs and macrophages. For example,suitable immunostimulatory agents for use in the present invention arecapable of stimulating APCs so that the maturation process of the APCsis accelerated, the proliferation of APCs is increased, and/or therecruitment or release of co-stimulatory molecules (e.g., CD80, CD86,ICAM-1, MHC molecules and CCR7) and pro-inflammatory cytokines (e.g.,IL-1β, IL-6, IL-12, IL-15, and IFN-γ) is upregulated. Suitableimmunostimulatory agents are also capable of increasing T cellproliferation. Such immunostimulatory agents include, but are not belimited to, CD40 ligand; cytokines, such as IFN-α, IFN-β, IFN-γ andIL-2; colony-stimulating factors, such as G-CSF (granulocytecolony-stimulating factor) and GM-CSF (granulocyte-macrophagecolony-stimulating factor); an anti-CTLA-4 antibody; toll receptoragonists (e.g., flagellin and MALP-2 (macrophage activatinglipopeptide-2); LPS (endotoxin); R837 (3M Pharmaceuticals, St. Paul,Minn.); R848 (3M Pharmaceuticals, St. Paul, Minn.); polyI:C(inosine:cytosine polynucleotide); ssRNA; dsRNA; Bacille Calmette-Guerin(BCG); Levamisole hydrochloride; and intravenous immune globulins.

As used herein, the term “linked” refers to the association of two ormore molecules. The linkage can be covalent or non-covalent. The linkagealso can be genetic (i.e., recombinantly fused). Such linkages can beachieved using a wide variety of art recognized techniques, such aschemical conjugation and recombinant protein production.

As used herein, the term antigen “cross-presentation” refers topresentation of exogenous protein antigens to T cells via MHC class Iand class II molecules on APCs.

As used herein, the term “T cell-mediated response” refers to anyresponse mediated by T cells, including effector T cells (e.g., CD8⁺cells) and helper T cells (e.g., CD4⁺ cells). T cell mediated responsesinclude, for example, T cell cytotoxicity and proliferation.

As used herein, the term “cytotoxic T lymphocyte (CTL) response” refersto an immune response induced by cytotoxic T cells. CTL responses aremediated primarily by CD8⁺ T cells.

As used herein, the term “antibody” includes whole antibodies orantigen-binding fragments thereof including, for example, Fab, F(ab′)₂,Fv and single chain Fv fragments. Suitable antibodies include any formof antibody, e.g., murine, human, chimeric, or humanized and any typeantibody isotype, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2,IgAsec, IgD, or IgE isotypes. As used herein, “isotype” refers to theantibody class that is encoded by heavy chain constant region genes.

Whole antibodies contain at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds. Each heavy chain is comprisedof a heavy chain variable region (abbreviated herein as HCVR or V_(H))and a heavy chain constant region. The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as LCVRor V_(L)) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The V_(H) and V_(L) regions canbe further subdivided into regions of hypervariability, termed“complementarity determining regions (CDR)”, interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

Preferred antibodies of the invention include human antibodies, e.g., ahuman antibody having an IgG1 (e.g., IgG1k) heavy chain and a kappalight chain. Other preferred antibodies of the invention bind human DCs,such as antibodies which bind a C-type lectin receptor on a human DC,e.g., the MR on human DCs. In a particular embodiment, the antibody is ahuman monoclonal antibody that binds to the human macrophage mannosereceptor (also referred to herein as “human B11 antigen”) having anapproximate molecular weight of 180 kD as measured by SDS-PAGE.Protocols for generating such antibodies are described in WO 01/085798,the contents of which are incorporated herein by reference. Particularhuman antibodies include those which comprise heavy and light chainvariable regions amino acid sequences as shown in SEQ ID NOs: 2 and 6,respectively, or an amino acid sequence that is sufficiently homologousto SEQ ID NO:2 or SEQ ID NO:6 such that the antibody retains the abilityto bind to dendritic cells

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., an antigen on a dendritic cell). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consistsof a VH domain; and (vi) an isolated complementarity determining region(CDR). Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. These antibody fragments are obtained using conventionaltechniques known to those with skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

The term “human antibody,” as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The terms “monoclonal antibody” or “monoclonal antibody composition,” asused herein, refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.Accordingly, the term “human monoclonal antibody” refers to antibodiesdisplaying a single binding specificity which have variable and constantregions derived from human germline immunoglobulin sequences. In oneembodiment, the human monoclonal antibodies are produced by a hybridomawhich includes a B cell obtained from a transgenic non-human animal,e.g., a transgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene, fused to an immortalized cell.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, (b) antibodies isolated from ahost cell transformed to express the antibody, e.g., from atransfectoma, (c) antibodies isolated from a recombinant, combinatorialhuman antibody library, and (d) antibodies prepared, expressed, createdor isolated by any other means that involve splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies can be subjected to in vitro mutagenesis(or, when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.

As used herein, “specific binding” refers to antibody binding to apredetermined antigen. Typically, the antibody binds with a dissociationconstant (K_(D)) of 10⁷ M or less, and binds to the predeterminedantigen with a K_(D) that is at least two-fold less than its K_(D) forbinding to a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen. The phrases “anantibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich binds specifically to an antigen.”

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) of 10⁻⁸ M or less, more preferably 10⁻⁹ M orless and even more preferably 10⁻¹⁰ M or less. However, “high affinity”binding can vary for other antibody isotypes. For example, “highaffinity” binding for an IgM isotype refers to an antibody having aK_(D) of 10⁻⁷ M or less, more preferably 10⁻⁸ M or less.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e., K_(d)/K_(a)) and is expressed as a molar concentration (M).

As used herein, the term “βhCG” refers to the beta subunit of humanchorionic gonadotropin and includes the whole antigen, antigenicfragments thereof, allelic variants thereof, and any polymorphisms,derived from the βhCG sequence (SEQ ID NO:20). βhCG is a hormonenecessary for the establishment of a successful pregnancy. Aside frompregnancy, the expression of this antigen is primarily restricted togerm cell tumors, as well as a significant number of adenocarcinomas.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule,” is used herein in referenceto nucleic acids encoding the molecular conjugates of the invention orportions thereof, e.g., SEQ ID NOs:9 and 11 or portions thereof, such asthe antigen or antibody portions (i.e., the V_(H), V_(L), or CDRs).Isolated nucleic acid molecules refer to a nucleic acid molecule inwhich the nucleotide sequences encoding the molecular conjugates arefree of other contaminating nucleotide sequences, e.g., a nucleotidesequence which does not encode any part of the molecular conjugate.

As disclosed and claimed herein, the sequences set forth in SEQ ID NOs:1-28 can include “conservative sequence modifications,” i.e., nucleotideand amino acid sequence modifications which do not significantly affector alter the functional characteristics of the molecular conjugate,e.g., the binding properties of the antibody portion of the construct orthe immunogenic properties of the antigen portion, encoded by thenucleotide sequence or containing the amino acid sequence. Suchconservative sequence modifications include nucleotide and amino acidsubstitutions, additions and deletions. Modifications can be introducedinto SEQ ID NOs: 1-28 by standard techniques known in the art, such assite-directed mutagenesis and PCR-mediated mutagenesis. Conservativeamino acid substitutions include ones in which the amino acid residue isreplaced with an amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a human anti-DCs antibodyis preferably replaced with another amino acid residue from the sameside chain family.

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a molecular conjugate coding sequence,such as by saturation mutagenesis, and the resulting modified molecularconjugates can be screened for appropriate functional activity.

Accordingly, molecular conjugates encoded by the nucleotide sequencesdisclosed herein and/or containing the amino acid sequences disclosedherein (i.e., SEQ ID NOs: 1-28) include substantially similar conjugatesencoded by or containing similar sequences which have beenconservatively modified. In particular, discussion as to howsubstantially similar antibodies can be generated for use in themolecular conjugates based on the partial (i.e., heavy and light chainvariable regions) sequences (SEQ ID NOs: 3, 4, 7, and 8) is providedbelow.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsodetermined using the algorithm of E. Meyers and W. Miller (Comput. Appl.Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences can be determined using the Needlemanand Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al, (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. Current Protocols in Molecular Biology, Greene Publishing andWiley Interscience, New York (1987).

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. Recombinant host cells include, for example, CHO cells andlymphocytic cells.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dog, cow,chickens, amphibians, reptiles, etc.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Antigens

Suitable antigens for use in the present invention include, for example,infectious disease antigens and tumor antigens, against which protectiveor therapeutic immune responses are desired, e.g., antigens expressed bya tumor cell or a pathogenic organism or infectious disease antigens.For example, suitable antigens include tumor-associated antigens for theprevention or treatment of cancers. Examples of tumor-associatedantigens include, but are not limited to, βhCG, gp100 or Pmel17,HER2/neu, CEA, gp100, MART1, TRP-2, melan-A, NY-ESO-1, MN (gp250),idiotype, MAGE-1, MAGE-3, Tyrosinase, Telomerase, MUC-1 antigens, andgerm cell derived tumor antigens. Tumor associated antigens also includethe blood group antigens, for example, Le^(a), Le^(b), LeX, LeY, H-2,B-1, B-2 antigens. Alternatively, more than one antigen can be includedwithin the antigen-antibody constructs of the invention. For example, aMAGE antigen can be combined with other antigens such as melanin A,tyrosinase, and gp100 along with adjuvants such as GM-CSF or IL-12, andlinked to an anti-APC antibody.

Other suitable antigens include viral antigens for the prevention ortreatment of viral diseases. Examples of viral antigens include, but arenot limited to, HIV-1 gag, HIV-1 env, HIV-1 nef, HBV core, FAS, HSV-1,HSV-2, p17, ORF2 and ORF3 antigens. Examples of bacterial antigensinclude, but are not limited to, Toxoplasma gondii or Treponemapallidum. The antibody-bacterial antigen conjugates of the invention canbe in the treatment or prevention of various bacterial diseases such asAnthrax, Botulism, Tetanus, Chlamydia, Cholera, Diptheria, Lyme Disease,Syphilis and Tuberculosis.

In a particular embodiment exemplified herein, the present inventionemploys an antigen comprising βhCG. This includes the entire βhCGsequence (SEQ ID NO:20) or any immunogenic (e.g., T cell epitopecontaining) portion of the sequence. As described below, suchimmunogenic portions can be identified using techniques known in the artfor mapping T cell epitopes, including algorithms and known T cellepitope mapping techniques. Examples of particular immunogenic peptidesfrom βhCG include those comprising SEQ ID NOs:21, 22, 23, 24, 25, 26,27, or 28, and conservative modifications thereof. Additionalimmunogenic peptides from βhCG, and methods for identifying suchpeptides, are described in U.S. Pat. Nos. 6,096,318 and 6,146,633, thecontents of which are incorporated by reference herein.

Antigenic peptides of proteins (i.e., those containing T cell epitopes)can be identified in a variety of manners well known in the art. Forexample, T cell epitopes can be predicted by analyzing the sequence ofthe protein using web-based predictive algorithms (BIMAS & SYFPEITHI) togenerate potential MHC class I and II-binding peptides that match aninternal database of 10,000 well characterized MHC binding peptidespreviously defined by CTLs. High scoring peptides can be ranked andselected as “interesting” on the basis of high affinity to a given MHCmolecule. As shown in FIG. 10 and using the sequence of the βhCG-B11conjugate (SEQ ID NO:10), both algorithms were used to identifyantigenic peptides from the βhCG portion (mustard) from which syntheticversions could be made and tested for their capacity to elicit T cellresponses in vitro. Thus, T cell epitopes were found for potentialbinding to HLA-A2, HLA-B7 and HLA-DR molecules. Several epitopes werealso predicted from the antibody (B11) segment of the βhCG-B11 conjugate(results not shown). Further, no T cell epitope was identified in the 37amino acid long C-terminal peptide (CTP).

Another method for identifying antigenic peptides containing T cellepitopes is by dividing the protein into non-overlapping peptides ofdesired length or overlapping peptides of desired lengths which can beproduced recombinantly, synthetically, or in certain limited situations,by chemical cleavage of the protein and tested for immunogenicproperties, e.g., eliciting a T cell response (i.e., proliferation orlymphokine secretion).

In order to determine precise T cell epitopes of the protein by, forexample, fine mapping techniques, a peptide having T cell stimulatingactivity and thus comprising at least one T cell epitope, as determinedby T cell biology techniques, can be modified by addition or deletion ofamino acid residues at either the amino or carboxy terminus of thepeptide and tested to determine a change in T cell reactivity to themodified peptide. If two or more peptides which share an area of overlapin the native protein sequence are found to have human T cellstimulating activity, as determined by T cell biology techniques,additional peptides can be produced comprising all or a portion of suchpeptides and these additional peptides can be tested by a similarprocedure. Following this technique, peptides are selected and producedrecombinantly or synthetically. Peptides are selected based on variousfactors, including the strength of the T cell response to the peptide(e.g., stimulation index). The physical and chemical properties of theseselected peptides (e.g., solubility, stability) can then be examined todetermine whether the peptides are suitable for use in therapeuticcompositions or whether the peptides require modification.

II. Antibody Vaccine Conjugates

The present invention provides a variety of therapeutic vaccineconjugates which include an antigen, such as a tumor or viral antigen,linked to an antibody that binds to an APC, e.g., via the mannosereceptor (MR). This allows for targeting of the antigen to APCs (e.g.,dendritic cells) to enhance processing, presentation and, ultimately, animmune response against the antigen(s), e.g., a CTL response.

In addition, the vaccine conjugate can include one or moreimmunostimulatory agents that also enhance the immune response againstthe antigen. Antibody-antigen vaccine conjugates of the invention can bemade genetically or chemically. In either case, the antibody portion ofthe conjugate may consist of the whole antibody or a portion of theantibody, such as the Fab fragment or single-chain Fv. In addition, morethan one antigen and/or immunostimulatory agent can be included in theconjugate.

In one embodiment, the vaccine conjugate comprises a human antibodyheavy chain that binds to human APCs and a human antibody light chainthat binds to human APCs, wherein either or both chains are linked tothe antigen and to an immunostimulatory agent. In another embodiment,the antigen and the immunostimulatory agent are separately linked toeither chain. In a particular embodiment, the antigen is βhCG.

Genetically constructed anti-dendritic antibody-antigen conjugates(e.g., those expressed as a single recombinant fusion protein) can bemade by linking an antigen of choice and/or an immunostimulatory agent(in the case of protein and peptide immunostimulatory agents) to theantibody at a variety of locations. Particular genetically producedconjugates (fusion constructs) of the invention include, for example,the βhCG-B11 construct, shown in FIG. 2. The βhCG-B11 constructcomprises human anti-dendritic cell antibody B11 fused to βhCG, atumor-associated antigen. The nucleotide sequence encoding thisconstruct is shown in SEQ ID NO:9.

For example, in one embodiment, the βhCG antigen and/or theimmunostimulatory agent can be fused to the end of the CH₃ domain of thehuman antibody heavy chain. The antigen and/or immunostimulatory agentalso can be fused at the hinged region of the antibody heavy chain inFab-fusion constructs, or in sequence with the variable light and heavychains (V_(H) and V_(L)) in single chain fusion constructs (ScFvconstructs). Alternatively, the antigen and/or immunostimulatory agentcan be fused to the antibody light chain instead of the antibody heavychain. Other points of fusion among the immunostimulatory agent, theantigen and the antibody can be used provided the genetic fusionconstruct can elicit a CTL response. A detailed map of the intactβhCG-B11 construct and the single chain B11 construct (pB11sfv-βhCG) areshown in Tables 1 and 2, respectively. Such genetic fusion conjugatescan include an antigen and an immunostimulatory agent linked to theantibody in either order (e.g., an antibody-antigen-immunostimulatoryconjugate or an antibody-immunostimulatory agent-antigen conjugate).

TABLE 1 βhCG-B11 Feature Map CDS (3 total)  BUsfr-bHCG   Start: 921 End:2153 neo   Start 3375 End: 4169 neomycin resistance gene  Amp   Start:5671 End: 6531 (Complementary) Ampicillin resistance gene Misc. Feature(5 total)  promoter   Start: 863 End: 882 promoter  signal sequence  Start 921 End: 977 B11 VL   Start: 978 End: 1296 B11VH   Start: 1344End: 1691 beta HCG   Start: 1712 End: 2164 PolyA Signal (2 total)  polyA   Start: 2267 End: 2491 poly A  poly A   Start: 4343 End: 4473 SV40poly A signal Promoter Eukaryotic (1 total)  promoter   Start: 232 End:819 eukaryotic promoter Promoter Prokaryotic (1 total)  promoter   Start6566 End: 6572 (Complementary) promoter Replication Origin (3 total) SV40 promoter and origin   Start 1 End: 1 origin of replication  F1origin   Start: 2537 End: 2965 origin of replication  pUC origin Start4856 End: 5526 (Complementary) origin

TABLE 2 pB11sfv-βhCG Feature Map CDS (4 total)  Light Chain   Start 735End: 1433 B11 Light Chain  C kappa   Start: 1113 End: 1433 AMP   Start:7810 End: 8670 (Complementary) amp   Original Location Description:complemented 1 ..6871)  DHFR   Start: 8921 End: 9484 dhfr   OriginalLocation Description: 7122-7685 Misc. Feature (9 total)  B11 VL   Start:792 End: 1112SV40 Promoter/Ori   Start 2298 End: 2622   SV40 promoterand origan of replication  Neo   Start: 2658 End: 3452 NeomicinResistance Gene  beta HCG   Start: 4015 End: 4467 (Complementary) bHCG CHS   Start: 4470 End: 4790 (Complementary) Heavy chain constant  region 3  CH2   Start: 4791 End: 5120 (Complementary) Heavy chainconstant   region 2  CH1   Start 5166 End: 5459 (Complementary) heavychain constant region 1  B11 VH   Start: 5460 End: 5807 (Complementary)Promoter   Start: 5905 End: 6559 (Complementary) PolyA Signal (3 total) Poly A   Start: 1526 End: 1757 PolyA   Start: 3744 End: 3975(Complementary) PolyA_Signal_2   Start 10282 End: 10411 SV40 poly A  Original Location Description: 8483..8612 Promoter Eukaryotic (1total)  Promoter   Start 9 End: 655

Chemically constructed antibody-antigen conjugates can be made using avariety of well known and readily available cross-linking reagents.These cross-linking reagents can be homofunctional or heterofunctionalcompounds, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),N-succinimidyl-S-acetyl-thioacetate (SATA), sulfosuccinimidyl4-(N-maleimidomethyl)cyclohaxane-1-carboxylate (sulfo-SMCC),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), that form covalent linkageswith different reactive amino acid or carbohydrate side chains on theanti-dendritic antibody and selected antigen. Other coupling andcross-linking agents also can be used to generate covalent linkages,such as protein A, carbodiimide, and o-phenylenedimaleimide (oPDM); (seee.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al.(1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include thosedescribed by Paulus (Behring Ins. Mitt. (1985) No. 78, 118-132); Brennanet al. (Science (1985) 229:81-83), and Glennie et al. (J. Immunol.(1987) 139: 2367-2375). Preferred conjugating agents are SATA andsulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).Immunostimulatory agents can also be chemically linked to the molecularconjugates of the present invention using the same linking methodsdescribed above.

Immunostimulatory agents and molecular conjugates can also be linked vianon-covalent methods, for example, using binding molecules such asstreptavidin and biotin. Other suitable binding molecules for use in thepresent invention are well-known in the art. In one aspect of theinvention, the nucleotide sequence encoding a streptavidin molecule isincorporated into the sequence encoding the molecular conjugate which,in turn, is linked to a biotinylated immunostimulatory agent.

Any antigen that can be cloned and expressed or purified can be selectedfor use in the present invention. Techniques for obtaining such antigensare well-known in the art. For example, tumor-associated antigens can bedirectly purified from cancer cells and identified by physiochemicaltechniques such as tandem mass spectrometry. Alternatively,tumor-specific T-cell clones can be tested against antigen-negativecells that have acquired antigen by being transfected with plasmid DNAclones to isolate the clone expressing the antigen. Synthetic peptidescan then be constructed to precisely identify the antigenic site orepitope.

As discussed above, the molecular vaccine conjugates of the inventioncan be administered together with, or include, one or moreimmunostimulatory agents. The immunostimulatory agent can beadministered separately or can be linked to the conjugate, eithercovalently, non-covalently, genetically, or a combination thereof,according to the linking techniques discussed above. Alternatively, theimmunostimulatory agent can be co-administered separately, for example,the agent can be administered simultaneously with the molecularconjugate, or prior to administration of the molecular conjugate, orsubsequent to administration of the molecular conjugate. A variety ofsuitable immunostimulatory agents are well known in the art and include,for example, CD40 ligand; cytokines, such as IFN-α, IFN-β, IFN-γ andIL-2; colony-stimulating factors, such as G-CSF (granulocytecolony-stimulating factor) and GM-CSF (granulocyte-macrophagecolony-stimulating factor); an anti-CTLA-4 antibody; toll receptoragonists (e.g., flagellin and MALP-2 (macrophage activatinglipopeptide-2); LPS (endotoxin); R837 (3M Pharmaceuticals, St. Paul,Minn.); R848 (3M Pharmaceuticals, St. Paul, Minn.); polyI:C(inosine:cytosine polynucleotide); ssRNA; dsRNA; Bacille Calmette-Guerin(BCG); Levamisole hydrochloride; and intravenous immune globulins.

In another aspect of the invention, partial antibody sequences from thevaccine construct can be used to express intact antibodies. Antibodies,such as the anti-APC antibodies (e.g., B11) encompassed by the vaccineconjugates of the present invention, interact with target antigens(e.g., C-type lectin receptors, such as the MR) predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature332:323-327; Jones, P. et al. (1986) Nature 321:522-525; and Queen, C.et al. (1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033). Suchframework sequences can be obtained from public DNA databases thatinclude germline antibody gene sequences. These germline sequences willdiffer from mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V(D)J joiningduring B cell maturation. Germline gene sequences will also differ fromthe sequences of a high affinity secondary repertoire antibody atindividual evenly across the variable region. For example, somaticmutations are relatively infrequent in the amino-terminal portion offramework region. For example, somatic mutations are relativelyinfrequent in the amino terminal portion of framework region 1 and inthe carboxy-terminal portion of framework region 4. Furthermore, manysomatic mutations do not significantly alter the binding properties ofthe antibody. For this reason, it is not necessary to obtain the entireDNA sequence of a particular antibody in order to recreate an intactrecombinant antibody having binding properties similar to those of theoriginal antibody (see WO 99/45962, which is herein incorporated byreferenced for all purposes). Partial heavy and light chain sequencespanning the CDR regions is typically sufficient for this purpose. Thepartial sequence is used to determine which germline variable andjoining gene segments contributed to the recombined antibody variablegenes. The germline sequence is then used to fill in missing portions ofthe variable regions. Heavy and light chain leader sequences are cleavedduring protein maturation and do not contribute to the properties of thefinal antibody. For this reason, it is necessary to use thecorresponding germline leader sequence for expression constructs. To addmissing sequences, cloned cDNA sequences can be combined with syntheticoligonucleotides by ligation or PCR amplification. Alternatively, theentire variable region can be synthesized as a set of short,overlapping, oligonucleotides and combined by PCR amplification tocreate an entirely synthetic variable region clone. This process hascertain advantages such as elimination or inclusion or particularrestriction sites, or optimization of particular codons.

The nucleotide sequences of heavy and light chain transcripts fromhybridomas are used to design an overlapping set of syntheticoligonucleotides to create synthetic V sequences with identical aminoacid coding capacities as the natural sequences. The synthetic heavy andkappa chain sequences can differ from the natural sequences in threeways: strings of repeated nucleotide bases are interrupted to facilitateoligonucleotide synthesis and PCR amplification; optimal translationinitiation sites are incorporated according to Kozak's rules (Kozak(1991) J. Biol. Chem. 266:19867-19870); and HindIII sites are engineeredupstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimizedcoding, and corresponding non-coding, strand sequences are broken downinto 30-50 nucleotide approximately the midpoint of the correspondingnon-coding oligonucleotide. Thus, for each chain, the oligonucleotidescan be assembled into overlapping double stranded sets that spansegments of 150-400 nucleotides. The pools are then used as templates toproduce PCR amplification products of 150-400 nucleotides. Typically, asingle variable region oligonucleotide set will be broken down into twopools which are separately amplified to generate two overlapping PCRproducts. These overlapping products are then combined by PCRamplification to form the complete variable region. It may also bedesirable to include an overlapping fragment of the heavy or light chainconstant region (including the BbsI site of the kappa light chain, orthe Agel site of the gamma heavy chain) in the PCR amplification togenerate fragments that can easily be cloned into the expression vectorconstructs.

The reconstructed heavy and light chain variable regions are thencombined with cloned promoter, translation initiation, constant region,3′ untranslated, polyadenylation, and transcription termination,sequences to form expression vector constructs. The heavy and lightchain expression constructs can be combined into a single vector,co-transfected, serially transfected, or separately transfected intohost cells which are then fused to form a host cell expressing bothchains.

Plasmids for use in construction of expression vectors for human IgGκare described below. The plasmids were constructed so that PCR amplifiedV heavy and V kappa light chain cDNA sequences could be used toreconstruct complete heavy and light chain minigenes. These plasmids canbe used to express completely human, or chimeric IgG₁κ or IgG₄κantibodies. Similar plasmids can be constructed for expression of otherheavy chain isotypes, or for expression of antibodies comprising lambdalight chains.

Thus, in another aspect of the invention, the structural features of theantibody portion of the vaccine conjugates described herein, e.g., B11,are used to create structurally related antibodies that retain at leastone functional property of the B11 antibody of the invention, such asbinding to APCs. More specifically, one or more CDR regions of B11 canbe combined recombinantly with known human framework regions and CDRs tocreate additional, recombinantly-engineered, anti-APC antibodies for usein the vaccine conjugates of the invention.

Accordingly, in another embodiment, the invention provides a method forpreparing a vaccine conjugate comprising an anti-DC antibody comprising:preparing an antibody comprising (1) human heavy chain framework regionsand human heavy chain CDRs, wherein at least one of the human heavychain CDRs comprises an amino acid sequence selected from the amino acidsequences of CDRs shown in FIG. 8 (SEQ ID NOs:13, 14, or 15); and (2)human light chain framework regions and human light chain CDRs, whereinat least one of the human light chain CDRs comprises an amino acidsequence selected from the amino acid sequences of CDRs shown in FIG. 9(SEQ ID NO:16, 17, or 18); wherein the antibody retains the ability tobind to APCs.

The ability of the antibody to bind APCs can be determined usingstandard binding assays, such as those set forth in the Examples (e.g.,an ELISA). Since it is well known in the art that antibody heavy andlight chain CDR3 domains play a particularly important role in thebinding specificity/affinity of an antibody for an antigen, therecombinant antibodies of the invention prepared as set forth abovepreferably comprise the heavy and light chain CDR3s of B11. Theantibodies further can comprise the CDR2s of B11. The antibodies furthercan comprise the CDR1s of B11. Accordingly, the invention furtherprovides anti-APC antibodies comprising: (1) human heavy chain frameworkregions, a human heavy chain CDR1 region, a human heavy chain CDR2region, and a human heavy chain CDR3 region, wherein the human heavychain CDR3 region is the CDR3 of B11 as shown in FIG. 8 (SEQ ID NO:15);and (2) human light chain framework regions, a human light chain CDR1region, a human light chain CDR2 region, and a human light chain CDR3region, wherein the human light chain CDR3 region is the CDR3 of B11 asshown in FIG. 9 (SEQ ID NO: 18), wherein the antibody binds DC. Theantibody may further comprise the heavy chain CDR2 and/or the lightchain CDR2 of B11. The antibody may further comprise the heavy chainCDR1 and/or the light chain CDR1 of B11.

Preferably, the CDR1, 2, and/or 3 of the engineered antibodies describedabove comprise the exact amino acid sequence(s) as those of B11disclosed herein. However, the ordinarily skilled artisan willappreciate that some deviation from the exact CDR sequences of B11 maybe possible while still retaining the ability of the antibody to bind DCeffectively (e.g., conservative substitutions). Accordingly, in anotherembodiment, the engineered antibody may be composed of one or more CDRsthat are, for example, at least 90%, 95%, 98% or 99.5% identical to oneor more CDRs of B11.

In addition or alternatively to simply binding APCs, engineeredantibodies such as those described above may be selected for theirretention of other functional properties of antibodies of the invention,such as:

(1) high affinity binding to APCs;

(2) binding to a unique epitope on an APC (to eliminate the possibilitythat monoclonal antibodies with complimentary activities when used incombination would compete for binding to the same epitope);

(3) induces a T cell-mediated immune response which is generated againstthe antigen; and/or

(4) induces a T cell response which comprises both CD4⁺ and CD8⁺ Tcell-mediated responses.

In another embodiment, a whole cell expressing the antigen of interest,e.g., βhCG, is transformed to express an anti-APC antibody, e.g., ananti-MR antibody, so that the antigen and the antibody are co-expressedby the cell. This can be done, for example, by transfecting the targetcell with a nucleic acid encoding a fusion protein containing atransmembrane domain and an anti-APC antibody. The cell expressing thevaccine conjugate can then be used to target APCs, e.g., DCs, to inducea CTL response.

Methods for generating such nucleic acids, fusion proteins, and cellsexpressing such fusion proteins are described, for example, in U.S.patent application Ser. No. 09/203,958, incorporated herein in itsentirety by this reference. Alternatively, the antibody can be bound toa cell or a pathogen by the use of chemical linkers, lipid tags, orother related methods (deKruif, J. et al. (2000) Nat. Med. 6:223-227;Nizard, P. et al. (1998) FEBS Lett. 433:83-88). Cells which express theantigen of interest and with surface-anchored antibodies may be used toinduce specific immune responses, e.g., a CTL response, against thecell, e.g., a tumor cell or microbial pathogen.

III. Pharmaceutical Compositions

In another aspect, the present invention provides therapeuticcompositions, e.g., pharmaceutical compositions, containing one or acombination of vaccine conjugates of the present invention formulatedtogether with an immunostimulatory agent. In one embodiment, theimmunostimulatory agent is linked to the vaccine conjugate. Thecompositions of the present invention may further include one or moreadjuvants and/or pharmaceutically acceptable carrier. The vaccineconjugate of the present invention is administered for delivery into thesubject's bloodstream for interaction with the subject's T cells. Suchtargeting of T cells can be accomplished either in vivo or ex vivo bydirectly using the conjugate or by using cells which have beenpreviously been targeted with vaccine conjugates.

The compositions of the present invention can additionally include othertherapeutic reagents, such as other antibodies, cytotoxins or drugs(e.g., immunosuppressants), and can be administered alone or incombination with other therapies, such as radiation. For example, avaccine conjugate that is rapidly internalized by APCs can be combinedwith a monoclonal antibody that enhances antigen presenting cellactivities of dendritic cells, e.g., release of immunostimulatorycytokines.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the vaccine conjugate may be coated in amaterial to protect the compound from the action of acids and othernatural conditions that may inactivate the compound.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

Compositions of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. The active compounds can be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Many methods for thepreparation of such formulations are patented or generally known tothose skilled in the art. See, e.g., Sustained and Controlled ReleaseDrug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978.

To administer a vaccine conjugate of the invention by certain routes ofadministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the compound may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the present inventioninclude those suitable for oral and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods known in the art of pharmacy. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the subject beingtreated, and the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compositionwhich produces a therapeutic effect. Generally, out of one hundredpercent, this amount will range from about 0.01 percent to aboutninety-nine percent of active ingredient, preferably from about 0.1percent to about 70 percent, most preferably from about 1 percent toabout 30 percent.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given alone or as apharmaceutical composition containing, for example, 0.01 to 99.5% (morepreferably, 0.1 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. In general, a suitabledaily dose of a compositions of the invention will be that amount of thecompound which is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. It is preferred that administration be intravenous,intramuscular, intraperitoneal, or subcutaneous, preferably administeredproximal to the site of the target. If desired, the effective daily doseof a therapeutic compositions may be administered as two, three, four,five, six or more sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms. While itis possible for a compound of the present invention to be administeredalone, it is preferable to administer the compound as a pharmaceuticalformulation (composition).

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carriercan be an isotonic buffered saline solution, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

When the active compound is suitably protected, as described above, thecompound may be orally administered, for example, with an inert diluentor an assimilable edible carrier.

IV. Uses and Methods of the Invention

Vaccine conjugates of the present invention can be used to treat and/orprevent (e.g., immunize against) a variety of diseases and conditions.

One of the primary disease indications is cancer. This includes, but isnot limited to, colon cancer, melanoma, lymphoma, prostate carcinoma,pancreatic carcinoma, bladder carcinoma, fibrosarcoma, rhabdomyosarcoma,mastocytoma, mammary adenocarcinoma, leukemia, or rheumatoidfibroblastsoma. Another primary disease indication is infectiousdiseases including, but not limited to, HIV, Hepatitis (e.g., A, B, &C), Influenza, Herpes, Giardia, Malaria, Leishmania, StaphylococcusAureus, Pseudomonas aeruginosa. Another primary disease indication isautoimmune diseases.

In a particular embodiment, the vaccine conjugates are used to treat orprevent diseases and conditions mediated by βhCG or cells expressingβhCG, which is a member of the cysteine-loop growth factor superfamily.Evidence suggests that βhCG plays a role in the establishment orprogression of cancers either as a growth factor, as an angiogenesisand/or metastasis-promoting agent, or as a suppressor of immune function(73). Accordingly, the present invention can be used to treat theprogression of cancers and other diseases involving angiogenesis. Theinvention also can be used to prevent or terminate unwanted pregnancy byinhibiting the role of βhCG and/or cells expressing βhCG in pregnancy.

For use in therapy, vaccine conjugates of the invention can beadministered to a subject directly (i.e., in vivo), either alone or withan immunostimulatory agent. In one aspect, the immunostimulatory agentis linked to the conjugate. Alternatively, the conjugates can beadministered to a subject indirectly by first contacting the conjugates(e.g., by culturing or incubating) with APCs, such as dendritic cells,and then administering the cells to the subject (i.e., ex vivo). Thecontacting and delivering of the conjugates to APCs, such that they areprocessed and presented by the APCs prior to administration, is alsoreferred to as antigen or cell “loading.” Techniques for loadingantigens to APCs are well known in the art and include, for example,Gunzer and Grabbe, Crit Rev Immunol 21 (1-3):133-45 (2001) and Steinman,Exp Hematol 24(8): 859-62 (1996).

In all cases, the vaccine conjugates and the immunostimulatory agentsare administered in an effective amount to exert their desiredtherapeutic effect. The term “effective amount” refers to that amountnecessary or sufficient to realize a desired biologic effect. Forexample, an effective amount could be that amount necessary to eliminatea tumor, cancer, or bacterial, viral or fungal infection. The effectiveamount for any particular application can vary depending on such factorsas the disease or condition being treated, the particular conjugatebeing administered, the size of the subject, or the severity of thedisease or condition. One of ordinary skill in the art can empiricallydetermine the effective amount of a particular multispecific moleculewithout necessitating undue experimentation.

Preferred routes of administration for the vaccine conjugates include,for example, injection (e.g., subcutaneous, intravenous, parenteral,intraperitoneal, intrathecal). The injection can be in a bolus or acontinuous infusion. Other routes of administration include oraladministration.

Vaccine conjugates of the invention also can be coadministered withadjuvants and other therapeutic agents, such as immunostimulatoryagents. The conjugates are typically formulated in a pharmaceuticallyacceptable carrier alone or in combination with such agents. Examples ofsuch carriers include solutions, solvents, dispersion media, delayagents, emulsions and the like. The use of such media forpharmaceutically active substances are well known in the art. Any otherconventional carrier suitable for use with the molecules falls withinthe scope of the instant invention.

Suitable agents for coadministration with the vaccine conjugates includeother antibodies, cytotoxins and/or drugs. In one embodiment, the agentis a anti-CTLA-4 antibody which are known to aid or induce immuneresponses. In another embodiment, the agent is a chemotherapeutic agent.The vaccine conjugates also can be administered in combination withradiation.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

EXAMPLES Methods and Materials

Generation of DCs from whole blood or leukopak: Human peripheral bloodmononuclear cells (PBMC) were obtained by density gradientcentrifugation of heparinized whole blood or apheresis preparations withFicoll-Paque. Monocytes were then isolated by adherence to plasticculture dishes or elutriation and differentiated into immature DCs byaddition of cytokines (10 ng/ml GM-CSF and 2 ng/ml IL-4) to the culturemedium. DCs were harvested between day 5 and 7 and analyzed by flowcytometry. The DCs prepared in this fashion were CD14⁻, HLA-DR⁺, CD11c⁺mannose receptor⁺ and expressed high levels of MHC Class I and II, CD80and CD86.

Selection of tumor antigen βhCG: βhCG is a subunit of human chorionicgonadotropin, a hormone necessary for the establishment of a successfulpregnancy. This glycoprotein subunit has a number of features that makeit an attractive antigen for cancer immunotherapy (reviewed in TriozziP. L. and Stevens V. (1999) Oncology Reports 6:7-17). First, aside frompregnancy, the expression of this antigen is primarily restricted togerm cell tumors, as well as a significant number of adenocarcinomas(Table 3). Also, hCG is a member of the cysteine-loop growth factorsuperfamily and may play a role in the a establishment or progression ofcancers either as a growth factor, an angiogenesis and/ormetastasis-promoting agent, or as a suppressor of immune function.Immunotherapy that limits the expression of functional hCG may thereforeoffer added therapeutic benefit.

TABLE 3 Percent of tumors positive for βhCG by immunohistochemistry(Triozzi P. L. and Stevens V. (1999)). Colon (52%) Lung (34%) Pancreas(31%) Esophagus (28%) Breast (24%) Bladder (21%) Ovary (19%) Cervix(18%) Gastric (18%)

Proliferation Assay: Effector T cells (5×10⁴) were co-cultured withautologous DCs (5×10³) loaded with or without antigen (MDX-1307 orother) in 96 well flat bottomed microplates in 0.2 ml final volume. Themixture was cocultured at 37° C. On day 4, cultures were pulsed with³H-thymidine (1 μCi/well) and 18 hours later, cells were harvesteddirectly on filters (Millipore). Filters were washed three times withwater followed by one wash in ethanol and allowed to dry under the hoodfor 5-10 min. Scintillation fluid (Packard, 20 μl/well) was then addedto the filters. Filter-bound radioactivity was determined by counting onthe Wallac beta counter. The results are expressed as stimulation index(S.I.) values in cpm of CTL stimulated with antigen versus stimulationwith no antigen or control antigen. For MHC blocking analysis, labeledtargets were preincubated with HLA-specific mAbs, W6/32 for blocking allclass I and L243 for blocking all class II HLA molecules (20 μg/ml), for30 min. at RT. Unbound mAb was removed by centrifugation.

Flow cytometry: Human DCs were prepared from monocytes by culture inGM-CSF and IL-4 for 5 days. DCs were incubated on ice with 10 μg/ml ofthe βhCG antigen/anti-MR antibody vaccine conjugate or an isotypecontrol. Vaccine conjugates were either directly FITC-labeled ordetected with an FITC-labeled anti-βhCG secondary monoclonal antibody.The cell associated fluorescence was determined using an LSR flowcytometer.

Cytotoxicity Assay: Target cells (3×10⁶), control and antigen loaded(βhCG-B11), were washed twice in RPMI medium and the pellet wasresuspended in 200 μl medium and labeled with 100 μCi ⁵¹Na₂CrO₄ for 60min at 37° C. Labeled targets were washed 3 times in RPMI medium and thepellet resuspended to yield a cell concentration of 3×10⁴ cells/ml.Antigen-specific CTL were titrated in a 96 well V-bottomed plate to giveratios of 100:1 (effector T cell, E: target, T) through to 12.5:1 orlower. A constant number of labeled targets were added (100 μl/well or3,000 target cells/well) and the plates were spun down at low speed(180×g) and incubated at 37° C. After 4 hours, 100-120 μl supernatantwas harvested and the radioactivity released was determined in aγ-counter counting (Wallac Instruments, Perkin-Elmer). CTL activity wascalculated and expressed as % Specific Lysis (killing) using thefollowing equation:

${{{Specific}\mspace{14mu} {Lysis}\mspace{14mu} (\%)} = {\frac{\begin{matrix}{{{Experimental}\mspace{14mu} {Release}\mspace{14mu} ({cpm})} -} \\{{Spontaneous}\mspace{14mu} {Release}\mspace{14mu} ({cpm})}\end{matrix}}{\begin{matrix}{{{Maximal}{\mspace{11mu} \;}{Release}\mspace{14mu} ({cpm})} -} \\{{Spontaneous}{\mspace{11mu} \;}{Release}\mspace{14mu} ({cpm})}\end{matrix}} \times 100}};$

where Experimental (cpm) refers to radioactivity (chromium released)from wells containing CTL (E) and target (T); Spontaneous (cpm) refersto the radioactivity from wells with targets in 0.1 ml medium alone(i.e. no CTL added) while Maximal release refers to radioactivity fromwells with targets in the presence of 0.1 ml detergent solution (IgepalCA 630; syn. NP-40; 5% solution in RPMI medium). Under well-controlledexperimental conditions, Spontaneous release values should be 10% ofMaximal release or less. For MHC blocking analysis, labeled targets werepreincubated with HLA-specific mAbs, W6/32 for blocking all class I andL243 for blocking all class II HLA molecules (20 μg/ml) for 30 min. atRT. Unbound mAb was removed by centrifugation and mAb-coated targetswere added to CTL. An isotype-matched mAb was used as a control.

Yet another way to look at cell-mediated immune responses is toinvestigate the proliferative capacity of antigen-driven T cells.Antigen-sensitized T cells tend to proliferate preferentially whenpreviously exposed antigens are presented in the context of MHC class IIand to a lesser extent, class I molecules. Thus, the enumeration ofdividing cells by uptake of a radioactive tracer provides a measure ofstimulation.

Example 1 Production of βhCG-B11

Design of vaccine conjugate: This construct was generated by linking theβhCG antigen to B11, a fully human antibody which binds to the humanmacrophage mannose receptor on dendritic cells. Linkage was accomplishedby covalently attaching the antigen to the heavy chain of the antibodyby way of a genetic fusion, as shown in FIG. 3.

Recombinant Expression of βhCG-B11 Vaccine Conjugate: As shown in FIG.2, a plasmid containing neomycin and dihydrofolate reductase genes wasgenerated containing the βhCG coding sequence fused to antibody B11 atthe CH₃ domain of the heavy chain (SEQ ID NOs:9 and 10). The resultingplasmid construct was transfected into CHO cells using a standardizedprotocol (Qiagen Inc, Valencia, Calif.). Transfected cells were selectedin media containing the antibiotic G418. Expression was furtheramplified by growing cells in increasingly higher concentrations ofmethatrexate. After amplification, the cells were cloned by limitingdilution, and stable clonal lines were used to generate cell banks forfurther studies. To confirm expression of the βhCG-B11 constructs,Western Blot analysis of proteins run on SDS-PAGE under reducingconditions was performed. This fusion protein was observed to be of theexpected molecular weight and to be properly assembled (i.e., to containboth the heavy chain fusion and the light chain). Specifically, thevaccine conjugate and the antibody alone were analyzed by SDS-PAGE usingdenaturing conditions and detected by Western blot analysis. The blotwas then probed separately using goat anti-human IgG heavy and light,and with a mAb (Sigma) specific to the βhCG C-terminal peptide. Theresults confirmed that the transformed CHO cells specifically expressedthe B11-βhCG vaccine conjugate as evidenced by the appropriate size andcomposition of the fusion product.

Example 2 Production of B11 scfv-βhCG

Design of vaccine conjugate: A second construct was generated by linkingthe βhCG antigen to a B11 single chain fusion (ScFv), which is a singlechain antibody that binds to the human macrophage mannose receptor ondendritic cells and contains the V_(L) and V_(H) fragments of the fullyhuman B11 antibody. Linkage was accomplished by covalently attaching theantigen to the carboxy terminus of the B11 ScFv by way of a geneticfusion, as shown in FIG. 1 (referred to as the B11sfv-hCG construct).

Recombinant Expression of B11sfv-βhCG Vaccine Conjugate: As shown inFIG. 1, a plasmid was generated containing the B11sfv-βhCG construct(SEQ ID NOs: 11 and 12). The resulting plasmid construct was transfectedinto mammalian cells using a standardized protocol (Qiagen Inc,Valencia, Calif.). Transfected cells were selected in media containingthe antibiotic G418. An ELISA was performed to confirm expression of theB11sfv-βhCG construct.

Example 3 Functional Characterization of Vaccine Conjugates

Antibody-targeted vaccine recognition of its cognate receptor on the APCsurface is the first step in this delivery platform. Flow cytometrystudies have been used to demonstrate that the βhCG-B11 and B11 sfv-βhCGconstructs bind specifically to cultured human DC expressing MR (FIG.4).

Using the anti-MR antibody as a probe, in situ staining of MR on humandermal DCs and macrophages in section of various human tissues wasexamined. Human tissue cryosections were stained with anti-MR humanantibody B11. DCs present in the dermal layer of the skin were clearlylabeled (data not shown) with the B11 antibody. It is noted that therewas binding to DCs in the dermal layer of skin. Furthermore,immunohistochemistry performed with the anti-MR B11 HuMAb staineddendritic cells in all tissues tested and showed no unexpectedcross-reactivity (results not shown). These studies have been repeatedwith the βhCG-B11 with identical results.

Example 4 Cross-Presentation of the βhCG Antigen/Anti-MR AntibodyVaccine Conjugate to T Cells

The capacity of the βhCG-B11 construct to be processed by DCs forpresentation of βhCG antigen to T cells via MHC class I and class IImolecules on DCs (cross-presentation) was evaluated. In particular, theβhCG-B11 construct was used to elicit antigen-specific T cells byculturing a pool of normal T cells with DCs that were exposed to thevaccine. The resulting “sensitized” T cells were then analyzed for theiractivity (proliferation and killing) and specificity. Specificity of theT cells can be demonstrated by comparing the T cell activity in responseto target cells that have the βhCG antigen to antigen-negative controls.Cytotoxic T cells (CTL), if present, should kill only those targets thatpresent βhCG related antigen but spare control targets that are eitherlacking the antigen or presenting an unrelated antigen. SinceCTL-mediated antigen recognition always occurs in the context of a givenMHC molecule bearing the peptide, blocking the MHC:peptide-CTLinteraction with an MHC-specific mAb confirms the class I or class IIpresentation.

Induction of antigen-specific effector T cells: Dendritic cells weregenerated from normal donor peripheral blood mononuclear cells (PBMC) byculturing adherent monocytes with 25 ng/ml recombinant human GM-CSF (R&Dsystems, MN) and 100 ng/ml of recombinant human IL-4 for 5 days. On day5, DCs were harvested (immature) and resuspended in AIM-V (serum-free)medium. The βhCG-B11 immunoconjugate (20 μg/ml) was added to 1.2×10⁶ DCand incubated for 45 min at 37° C. Antigen-loaded DCs was allowed tomature in the presence of CD40L (Peprotech, N.J.; 20 ng/ml) for at least24 hours. Mature DC (1×10⁶) were washed once and added to T cells(2×10⁷; bulk) previously seeded in 24 well plates at 1×10⁶ cells/ml(ratio of DC: T cells, 20). The following culture conditions wereemployed: addition of 10 ng/ml IL-7 on day 0, followed by 10 ng/ml ofIL-10 on day 1 (at 24 hours), and 20 U/ml IL-2 on day 2 (at 48 hours).Restimulation was carried out on days 7, 14 and 21 as before, exceptthat βhCG-B11 concentration was cut by half (10, 5 and 2.5 μg/ml,respectively). T cells were tested for reactivity (either in bulk orwith purified T cell sub populations) against ⁵¹Cr-labeled DC loadedwith nothing, βhCG-B11, B11 sfv-βhCG, or B11. MHC-specificity wasascertained in the presence of HLA-specific mAbs.

As illustrated in FIG. 5, the βhCG-B11 construct induced βhCG-specificcytotoxic T cells. No killing ensued if the T cells were cultured withtargets that do not present βhCG. The target cells used in theseexperiments were HLA-matched DC treated with the βhCG-B11 construct orcontrol antigens. Target cells treated only with the anti-MR antibody(B11) were not susceptible to the cytotoxic activity, demonstrating thatonly the antigen portion of the vaccine was able to elicit CTL activity.These results show that the βhCG-B11 construct induces efficient CTLactivity and, specifically, the CTL activity is directed towards theβhCG antigen but not the targeting antibody (B11).

Furthermore, the potent killing of targets presenting βhCG antigen wasreproduced with purified CD8⁺ T cells, which killing was blocked in thepresence of anti-MHC class I antibodies (FIG. 6). In particular, theβhCG-B11 construct was used to generate βhCG-specific T cells fromperipheral blood mononuclear cells of two donors. CD8⁺ and CD4⁺ T cellswere purified from bulk cultures using immunomagnetic beads.Cytotoxicity assays were carried out as described above with theeffector:target ratio set at 40:1. The target cells (immature DC) wereuntreated (control) or loaded with the βhCG-B11 construct. Todemonstrate MHC Class I specificity, target cell killing was blocked bypreincubation with an HLA-specific antibody (W6/32).

Collectively, these data (FIGS. 6 and 7) confirm the ability of theβhCG-B11 construct to induce potent βhCG-specific CTL, and additionallydemonstrate that the CTL activity is mediated by CD8⁺ T cells in anHLA-dependent manner. No killing activity was observed with the purifiedCD4⁺ T cells.

As shown in FIG. 7, the βhCG-B11 construct-elicited T cells proliferatein response to the βhCG-B11 construct targeted DC. In particular, DCwere treated with the βhCG-B11 construct to generate βhCG-specific Tcells from peripheral blood mononuclear cells. T cells from bulkcultures (CD4⁺ and CD8⁺ T cells) were tested for proliferation inresponse to antigen stimulation. T cells were co-cultured with untreatedDC (control) or DC loaded with the βhCG-B11 construct with or withoutHLA blocking antibodies. To measure proliferation, DNA synthesis wasanalyzed after 5 days of culture using ³H-thymidine. The data wereexpressed as the fold-increase in proliferation (stimulation index) overcontrol. As seen with the CTL activity, no appreciable response wasfound when the T cells were stimulated by DC alone (i.e., no antigen).DC targeted with only the unconjugated antibody (anti-MR B11 mAb) didnot induce proliferation of T cells elicited by the βhCG-B11 construct.The proliferative capacity of the T cells was significantly blocked inthe presence of both anti-MHC class I as well as class II-specific mAbs,demonstrating that both CD4⁺ and CD8⁺ T cells were responding. Thesedata show that the uptake of the βhCG-B11 construct by DC enables thevaccine to gain access to MHC class I and class II processing pathways,which is consistent with co-localization of MR with MHC compartments.

Example 5 Internalization by DCs of Anti-MR Antibody B11 vs.Internalization by DCs of a Mannosylated Antigen (Inhibition of ClathrinMediated Internalization)

Immature DCs can take up soluble antigens by pinocytic or receptormediated endocytic mechanisms (55). The mechanism of antigeninternalization determines its intracellular fate and may effect thequality of immune response to it (54, 55, 56). Internalization throughthe MR has been described as a rapid, clathrin mediated internalizationevent (57, 58). The MR itself has two putative clathrin targetingsequences within its cytoplasmic tail, and internalization ofmannosylated gold particles have localized to clathrin-coated pits by EM(58, 59). Clathrin dependant endocytosis can be specifically disruptedby brief hypertonic shock or K+depletion (61).

In order to determine if mannosylated antigens or B11 bound to themannose receptor were internalized via clathrin-coated pits, immatureDCs were incubated on ice in AIM5 media with or without 400 mM sucrosefor 30 min in the presence of either B11 mAb or mannosylated BSA. Cellswere then warmed to 37° C. and allowed to internalize for 20 minutes.After being washed and fixed, cells were analyzed by confocal microscopy(data not shown). When B11 was bound to the MR, its uptake was inhibitedby hypertonic shock, indicating that its mechanism of internalizationwas through clathrin coated-pits. Uptake of mannosylated BSA, incontrast, was not inhibited by hypertonic shock, indicating that itsmechanism of internalization was not dependent on clathrin coated-pitformation. Even at concentration 20 fold higher than that of B11,surface staining by mannosylated BSA FITC was relatively weak.Subsequent studies revealed that internalized mannosylated BSA FITCco-localized with non-specific, fluid phase tracers, where as vesiclescontaining internalized B11 excluded the non-specific tracer (data notshown). In contrast to B11-FITC the uptake of both mannosylated BSA-FITCand the fluid phase tracer was largely blocked by pretreatment with thePI3K inhibitor wortmannin (data not shown). These results indicate thatthe vast majority of mannosylated BSA was taken up by the immaturedendritic cell was through non-specific macropinocytic mechanisms,suggesting that the quality of immune response to the mannosylatedantigen may differ greatly from antigen specifically targeted to the MR.

Example 6 Binding of B11sfv-βhCG to DCs

Monocyte-derived DCs were exposed either to B11 sfv-βhCG or βhCG-B11 inPBS-BSA buffer for 45 minutes at 37° C. and allowed to mature overnightin the presence of CD40L. Harvested DCs were then washed and stainedwith mouse anti-βhCG followed by goat anti-hu IgG (F_(c))-PE conjugate.Stained cells were analyzed on a flow cytometer (BD-LSR). Approximately,10,000 events were collected for each sample. Backgroundautofluorescence and isotype matched antibody staining served ascontrols. Based on the mean fluorescence intensity (MFI) (data notshown), B11 sfv-βhCG binding to MR expressed on DC is similar to that ofβhCG-B11.

Example 7 CTLs Specific for the βhCG-B11 Construct Recognize the scFvForm of the Antigen (B11sfv-βhCG) Presented by DCs

CTL raised to DC-presented βhCG-B11 were tested against autologous DCtargets that were exposed to βhCG-B11 and B11 sfv-βhCG, while untreatedDC or DC exposed to B11 served as controls. Following antigen exposure,targets were labeled with ⁵¹chromium and mixed with CTL in a 4 hourassay that measures release of radioactivity in the supernatant. In thisexperiment, βhCG-B11-specific T cells recognize two of four targets thatpresent the antigen on MHC class I molecules. No killing of targetsensues when DC lack antigen (FIG. 11). Thus, the uptake of βhCG-B11 byDC likely results in a βhCG-derived T cell epitope recognized by CTL.

EQUIVALENTS

Those skilled in the art will recognize or be able to ascertain, usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Incorporation by Reference

All patents, pending patent applications and other publications citedherein are hereby incorporated by reference in their entirety.

REFERENCES

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1. A method of inducing or enhancing a T cell mediated immune responseagainst an antigen comprising contacting antigen presenting cells (APCs)with (a) a molecular conjugate comprising an antigen and a monoclonalantibody which binds to the human macrophage mannose receptor (MMR); and(b) a toll receptor agonist or a combination of toll receptor agonists,such that the antigen is internalized, processed and presented to Tcells in a manner which induces or enhances a T cell mediated immuneresponse against an antigen.
 2. The method of claim 1, wherein the tollreceptor agonist is selected from the group consisting of flagellin,MALP-2 (macrophage activating lipopeptide-2), LPS, R837, R848, polyI:C(inosine:cytosine polynucleotide), ssRNA, and dsRNA.
 3. The method ofclaim 1, wherein the combination of toll receptor agonists comprisesR848 and polyI:C.
 4. The method of claim 1, wherein the antigen is abacterial, viral or tumor antigen.
 5. The method of claim 4, wherein theantigen is a tumor-associated antigen selected from βhCG, gp100 orPmel17, HER2/neu, CEA, gp100, MART1, TRP-2, melan-A, NY-ESO-1, MN(gp250), MAGE-1, MAGE-3, Tyrosinase, Telomerase, and MUC-1 antigens. 6.The method of claim 4, wherein the antigen is a viral antigen selectedfrom HIV-1 gag, HIV-1 env, HIV-1 nef, HBV core, FAS, HSV-1, HSV-2, p17,ORF2 and ORF3 antigens.
 7. The method of claim 4, wherein the antigen isa bacterial antigen selected from the group consisting from Toxoplasmagondii, Treponema pallidum, Anthrax, Botulism, Tetanus, Chlamydia,Cholera, Diptheria, Lyme Disease, Syphilis, and Tuberculosis antigens.8. The method of claim 5, wherein the antigen is a βhCG antigen.
 9. Themethod of claim 1, wherein the APCs are dendritic cells.
 10. The methodof claim 1, wherein the T cell response is induced through both MHCClass I and MHC Class II pathways.
 11. The method of claim 1, whereinthe T cell response is mediated by cytotoxic T cells and/or helper Tcells.
 12. The method of claim 1, wherein the T cell response is inducedby cross-presentation of the antigen to T cells through both MHC Class Iand MHC Class II pathways.
 13. The method of claim 1, wherein theantibody is selected from the group consisting of human, humanized andchimeric antibodies.
 14. The method of claim 1, wherein the antibody isselected from the group consisting of a whole antibody, an Fab fragmentand a single chain antibody.
 15. The method of claim 1, wherein theantibody comprises a heavy chain variable region comprising FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4 sequences and a light chain variable regioncomprising FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 sequences, wherein:(a) the heavy chain variable region CDR3 sequence comprises SEQ ID NO:15; and (b) the light chain variable region CDR3 sequence comprises SEQID NO: 18; (c) the heavy chain variable region CDR2 sequence comprisesSEQ ID NO: 14; (d) the light chain variable region CDR2 sequencecomprises SEQ ID NO: 17; (e) the heavy chain variable region CDR1sequence comprises SEQ ID NO:13; and (f) the light chain variable regionCDR1 sequence comprises SEQ ID NO:
 16. 16. The method of claim 1,wherein the antibody comprises a heavy chain variable region comprisingthe amino acid sequence shown in SEQ ID NO:4 and a light chain variableregion comprising the amino acid sequence shown in SEQ ID NO:8, or anantibody that competes for binding with the foregoing antibody.
 17. Themethod of claim 1, wherein the molecular conjugate and the toll receptoragonist or combination of toll receptor agonists, are linked.
 18. Themethod of claim 17, wherein the molecular conjugate and thetoll-receptor agonist are expressed together as a single recombinantfusion protein.
 19. The method of claim 1, further comprisingadministration of a colony-stimulating factor.
 20. The method of claim1, wherein the toll receptor agonist or combination of toll receptoragonists, is administered simultaneously with the molecular conjugate.21. The method of claim 1, wherein the toll receptor agonist orcombination of toll receptor agonists, is administered prior toadministration of the molecular conjugate.
 22. The method of claim 1,wherein the toll receptor agonist or combination of toll receptoragonists, is administered subsequent to administration of the molecularconjugate.